Low voltage-activated Ca 2ϩ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, ␣1I or Ca v T.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels ␣1G and ␣1H and to the high voltage-activated channels formed by ␣1E␤ 3 . The ␣1I channels opened after small depolarizations of the membrane similar to ␣1G and ␣1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca 2ϩ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The ␣1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba 2ϩ solutions. Based on its brain distribution and novel gating properties, we suggest that ␣1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

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Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.

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Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl2 and 5.4 mM KCl (35 degrees C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50 +/- 5 pF per cell, an input resistance of 200 +/- 25 kOhm X cm2 and a series resistance of 44 +/- 4 Ohm X cm2. The cells had resting potentials of -52 +/- 2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (-38 mV) with a maximal rate of 6.5 +/- 1.8 V/s to an overshoot of 22 +/- 3 mV. The AP lasted for 36 +/- 4 ms (measured between threshold and -40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (iCa, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward current iK. Ca-channel blockers (5 mM NiCl2) reduced iK suggesting that (part of) iK was Ca-activated. iK rose within about 100 ms to a peak of 40-200 muA/cm2 from which it inactivated slowly and incompletely. The inactivating iK followed a bell-shaped voltage-dependence, the noninactivating iK an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at -36 mV and a slope of 9 mV. Repolarization to -50 mV induced outward tail currents which reversed polarity at -85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion to iK during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse. iK was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

L-type Ca 2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intraceUular pH (pHi). Whole cell lca fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36°C), and increased when pHi was made more alkaline by addition of 20 mM NH4C1. Peak/Ca was less pHi sensitive than late/ca (170 ms after depolarization to 0 mV). pHi-effects on single Ca ~+ channel currents were studied with 110 mM BaCI2 as the charge carrier (22°C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4CI or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re-openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca 2+ homeostases and vasotonus is discussed.

Voltage-operated Ca2+ channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of alpha1 subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the alpha1E subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II-III loop), and the carboxy terminus. Mouse and human alpha1E transcripts have been analysed by reverse transcription-polymerase chain reaction and by sequencing of amplified fragments. For the II-III loop three different alpha1E cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted alpha1E cDNA fragments of the carboxy terminus are identified in vivo. Two different alpha1E constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild-type alpha1E subunit showed properties, typical for a high-voltage activated Ca2+ channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different alpha1E isoforms changes during development. In human cerebellum the longer alpha1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two alpha1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.

The expression of Ca 2ϩ channel A1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned A1E cDNA sequences led to the prediction of A1E splice variants. Transcripts of two cloned A1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter A1E isoform have been assigned to the rat cerebrum and to neuron-like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)-PCR and visualized on the protein level by Western blotting with common and isoformspecific antibodies. Transcripts of the longer A1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS-1 (rat) and βTC-3 (mouse), in the pituitary cell line AtT-20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of A1E in cell lines and tissues shows that the wide expression of A1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called A1Ee has been determined by RT-PCR, while in cerebellum a set of different A1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue-specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform A1Ee in islets of Langerhans and kidney leads to the suggestion that A1E might be involved in the modulation of the Ca 2ϩ -mediated hormone secretion.Keywords : A1E isoforms ; Ca 2ϩ channels ; islets of Langerhans; kidney; single-cell RT-PCR.Low and high voltage-gated Ca 2ϩ channels are subdivided, according to their threshold of activation and their different single channel conductances [1]. The pore-forming subunit of the first low voltage-activated T-type Ca 2ϩ channel has been cloned and functionally expressed recently as A1G [2].The group of six different high voltage-activated Ca 2ϩ channels (A1S-, A1C-, A1D-, A1A-, A1B-, A1E subunits) has been investigated intensively during the last decade [3,4]. They are subdivided into two different families, based on their primary sequence. The A1S, A1C, and A1D subunits are sensitive against the classical blockers of L-type Ca 2ϩ channels, the dihydropyridines, the phenylalkylamines and the benzothiazepines. The subfamily of the non-L-type A1 subunits share, among each other, a functional interaction with inhibitory G proteins [5]. The Ntype Ca 2ϩ channels (A1B) are blocked by ω-conotoxin-GVIA, the P-type and Q-type Ca 2ϩ channels (A1A) are discriminated byCorrespondence to T. Schneider,